PROJECT SUMMARY Project title: RNA metabolism mediated by the Integrator complex Precise recognition of RNAs by RNA-binding proteins and RNA-specific enzymes is critical for gene expression and cell growth. In recent years, the Integrator protein complex has emerged as an essential component in RNA metabolism. Disrupted RNA regulation caused by a malfunctioned Integrator has been implicated in multiple malignancies, including developmental failure (malformations of cortical development), neurodegenerative disease (Cornelia de Lange syndrome) and cancer (lung and prostate carcinomas). Yet, knowledge about the underlying pathological mechanisms is lacking. This is largely due to a limited understanding of the interplay between Integrator and its RNA substrates. We and others found that Integrator is an RNA-specific enzyme that processes several species of regulatory non-coding RNA (ncRNA). Furthermore, Integrator regulates the generation of mRNAs that are rapidly activated in response to growth factor stimulation. How Integrator recognizes RNAs through one or more of its 14 protein subunits is unclear. In the next five years, by developing new biochemical techniques, we will purify from human cells the key Integrator protein subunits that mediate RNA recognition and binding. Subsequently, we will use these RNA-recognition subunits to identify the RNAs that directly bind to, and are processed by, the Integrator complex. Our recent data suggest that one of the potential RNAs processed by Integrator are a specific set of microRNAs, small ncRNA molecules that control the function of other genes and hence are important for controlling human developmental and disease processes. microRNAs that are processed by Integrator are special in that they are produced in cells where most of the conventionally-generated microRNAs are not present. Several of these microRNAs function to suppress the growth of tumors. We will investigate how these microRNAs are uniquely generated and how they regulate the expression of other genes. There is relatively little known about the function of Integrator, how it controls RNA metabolism and how these RNAs operate within cells. Therefore, our research fills a major gap in knowledge. Our efforts in the next five years will not only uncover a new class of RNA-binding proteins as important gene regulators, but will also provide the basis for developing therapeutics to combat diseases that are caused by Integrator deficiency and dysregulated microRNAs. Techniques and experience acquired in this project will prepare our group for future research in exploring molecular machineries that generate and regulate RNAs essential for gene expression.